Recombinant Protein Production Introduction to Expression Systems Core Facility of Recombinant Protein Production, National Research Program for Genomic Medicine

Recombinant Protein Production -Why? over-expression to get enough amount easy purification -Application functional studies structural studies vaccine/antigen/antibodies therapeutic drug industrial enzymes for reaction

Application: Drug Discovery

Application: therapeutic proteins Actimmune (If g) Activase (TPA) BeneFix (F IX) Betaseron (If b) Humulin Novolin Pegademase (AD) Epogen Regranex (PDGF) Novoseven (F VIIa) Intron-A Neupogen Pulmozyme Infergen Now more than 200 approved peptide and protein pharmaceuticals on the FDA list (http://www.accessexcellence.org/RC/AB/IWT/The_Biopharmaceuticals.html)

Application: structural genomics Bioinformatics

Principle in Protein Production Bioinformatics Target identification and cloning Protein expression test Protein purification and production Applications

Protein Expression Bottleneck Protein Biochemistry soluble, purifiable protein Enzymology soluble, active protein 0.1-10 mg of protein Crystallography soluble, crystallizable protein 5-100 mg of protein

Bottlenecks to efficient protein expression in E. coli Inefficient transcription No or little protein synthesized u Promoter choice and design l Inefficient translation No or little protein synthesized u Codon usage Transcript stability Transcript secondary structure u u l Inefficient folding (cytoplasmic or periplasmic) Aggregation or degradation u Improper secondary, tertiary or quaternary structure formation Inefficient or improper disulfide bridge formation Inefficient isomerization of peptidyl-prolyl bonds u u l Inefficient membrane insertion/translocation Aggregation or degradation l Toxicity Cell death

Protein Expression and Purification Isolation of gene of interest Introduction of gene to expression vector Transformation into host cells Growth of cells through fermentation Isolation & purification of protein

+ Cloning and expression of target gene: Expression of Fusion Protein Gene of Interest Recombinant Vector Expression Vector Expression of Fusion Protein

Cloning Process Gene of interest is cut out with restriction enzymes (RE) Host plasmid (circular chromosome) is cut with same REs Gene is inserted into plasmid and ligated with ligase New (engineered) plasmid inserted into bacterium (transform)

Cloning (Details)

Cloning (Details) protein

Recombinant Protein Expression Systems Escherichia coli Other bacteria Pichia pastoris Other yeast Baculovirus Animal cell culture Plants Sheep/cows/humans Cell free Polyhedra

Expression System Selection Choice depends on size and character of protein Large proteins (>100 kD)? Choose eukaryote Small proteins (<30 kD)? Choose prokaryote Glycosylation essential? Choose baculovirus or mammalian cell culture High yields, low cost? Choose E. coli Post-translational modifications essential? Choose yeast, baculovirus or other eukaryote

Which Vector? Must be compatible with host cell system (prokaryotic vectors for prokaryotic cells, eukaryotic vectors for eukaryotic cells) Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site

Plasmids and Vectors Circular pieces of DNA ranging in size from 1000 to 10,000 bases Able to independently replicate and typically code for 1-10 genes Often derived from bacterial “mini” chromosomes (used in bacterial sex) May exist as single copies or dozens of copies (often used to transfer antibiotic resistance)

Key Parts to a Vector Origin of replication (ORI) – DNA sequence for DNA polymerase to replicate the plasmid Selectable marker (Amp or Tet) – a gene, when expressed on plasmid will allow host cells to survive Inducible promoter – Short DNA sequence which enhances expression of adjacent gene Multi-cloning site (MCS) – Short DNA sequence that contains many restriction enzyme sites

A Generic Vector

Which Vector? Promoters arabinose systems (pBAD), phage T7 (pET), Trc/Tac promoters, phage lambda PL or PR Tags His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coil/K-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags

Gene Introduction (Bacteria)

Bacterial Transformation

Bacterial Transformation Moves the plasmid into bacterial host Essential to making the gene “actively” express the protein inside the cell 2 routes of transformation CaCl2 + cold shock Electroporation Typical transformation rate is 1 in 10,000 cells (not very efficient) for CaCl2, but 1 in 100 for electroporation

Electroporator 25 microfarads = 2500 V @ 200 ohms for 5 ms

Electroporation Seems to cause disruption in cell membrane Reconstitution of membrane leads to large pores which allow DNA molecules to enter Works for bacteria, yeast and animal cells

Bacterial Systems Advantages Disadvantages Grow quickly (8 hrs to produce protein) High yields (50-500 mg/L) Low cost of media (simple media constituents) Low fermentor costs Difficulty expressing large proteins (>50 kD) No glycosylation or signal peptide removal Eukaryotic proteins are sometimes toxic Can’t handle S-S rich proteins

Cloning & Transforming in Yeast Cells Pichia pastoris

Pichia Pastoris Yeast are single celled eukaryotes Behave like bacteria, but have key advantages of eukaryotes P. pastoris is a methylotrophic yeast that can use methanol as its sole carbon source (using alcohol oxidase) Has a very strong promoter for the alcohol oxidase (AOX) gene (~30% of protein produced when induced)

Pichia Cloning

Pichia Pastoris Cloning Uses a special plasmid that works both in E. coli and Yeast Once gene of interest is inserted into this plasmid, it must be linearized (cut open so it isn’t circular) Double cross-over recombination event occurs to cause the gene of interest to insert directly into P. pastoris chromosome where the old AOX gene used to be Now gene of interest is under control of the powerful AOX promoter

Pichia Systems Advantages More advantages Grow quickly (8 hrs to produce protein) Very high yields (50-5000 mg/L) Low cost of media (simple media constituents) Low fermentor costs Can express large proteins (>50 kD) Glycosylation & signal peptide removal Has chaperonins to help fold “tough” prtns Can handle S-S rich proteins

Baculovirus Expression

Baculovirus Expression Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs) Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae) Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell

Baculovirus Expression ~12 days

Baculovirus (AcMNPV) Cloning Process 5’ 3’ Transfer vector Polyhedrin gene x Cloned gene Cloned gene 5’ 3’ AcMNPV DNA Recombinant AcMNPV DNA

Baculovirus Systems Disadvantages Advantages Grow very slowly (10-12 days for set-up) Cell culture is only sustainable for 4-5 days Set-up is time consuming, not as simple as yeast Can express large proteins (>50 kD) Correct glycosylation & signal peptide removal Has chaperonins to help fold “tough” prtns Very high yields, cheap

Mammalian Expression Systems

Mammalian Cell-line Expression Sometimes required for difficult-to-express proteins or for “complete authenticity” (matching glycosylation and sequence) Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line Vectors usually use SV-40 virus, CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene

Mammalian Expression Gene initially cloned and plasmid propagated in bacterial cells Mammalian cells transformed by electroporation (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression & integration of DHFR and the gene of interest

Methotrexate (MTX) Selection Gene of interest DHFR Transfect dfhr- cells Grow in Nucleoside Free medium Culture a Colony of cells Grow in 0.05 uM Mtx Culture a Colony of cells

Methotrexate (MTX) Selection Grow in 0.25 uM Mtx Culture a Colony of cells Grow in 5.0 uM Mtx Culture a Colony of cells Foreign gene expressed in high level in CHO cells

Mammalian Systems Disadvantages Advantages Selection takes time (weeks for set-up) Cell culture is only sustainable for limited period of time Set-up is very time consuming, costly, modest yields Can express large proteins (>50 kD) Correct glycosylation & signal peptide removal, generates authentic proteins Has chaperonins to help fold “tough” prtns

Conclusion Isolation of gene of interest Introduction of gene to expression vector Transformation into host cells Growth of cells through fermentation Isolation & purification of protein

National Research Program for Genomic Medicine Core Facility of Recombinant Protein Production重組蛋白質生產核心設施 D1

Expression systems E. coli Baculovirus Yeast Cell-free Mammalian cell ( not open for service)

Expresssion Systems SYSTEMS Advantages Disadvantages E. coli Parallel cloning Fast Ease of use Low cost Poor expression Low solubility Lacking post-translational modifications Cell-free Faster Skips cell transformation, growing, and lysis Low protein yield Expensive Tricky to optimize the lysate and expression conditions Yeast Glycosylation Efficient Economical Protein with disulfide bonds Different glycosylation to mammalian cells Baculovirus Most proper eukaryotic Duration of expression limited to infection period Virus production contains numerous steps Maintain high virus titers Mammalian cells Native environment for mammalian proteins Lower protein yield

E. coli - the most popular expression system

E. coli Expression System -challenge poorly expressed protein insoluble- inclusion bodies expressed and soluble: 20-30% -improvement growth condition (e.g. temperature) codon usage host strain fusion to carrier protein

parallel screening for soluble proteins E. Coli Expression System parallel screening for soluble proteins Rationale 1. Increase the expression level and solubility of target protein with protein tags. 2. Simultaneously, parallel screening different fusion tags. 3. Has potential for automating gene cloning. Publication Protein Science (2002), Shih YP et. al., 11:1714-1719.

E. coli Sticky-end PCR

E. coli Parallel Gene Cloning

Parallel screening for soluble protein E. coli Parallel screening for soluble protein

Statistical analysis of soluble protein ratio E. coli Statistical analysis of soluble protein ratio

E. coli Expression System - Modified version EcoR I Xho I Promotor Fusion tag Thrombin His*6 FXa Target Protein Terminator To improve consistency and convenience, we now modify the above vectors to include a hexa-His tag and a Factor Xa cleavage site at the N-terminus of each protein expressed in E. coli EcoR I Xho I Promotor Fusion tag Thrombin His*6 FXa Target Protein Terminator

技術比較說明 融合蛋白質的選擇類似，主要是cloning的差別 E. coli 技術比較說明 融合蛋白質的選擇類似，主要是cloning的差別 Hammarström et al. Protein Science (2002), 11:313–321 他人已使用商品化的策略；Gateway Technology (Invitrogen) Donor vector Purify plasmid PCR Ligation Co-transformation 我們使用Sticky-end PCR的方法，不必經過Sub-cloning即可parallel cloning PCR Denature Re-nature

E. Coli Expression System Summary The method introduces sticky-end to target genes, without using restriction enzymes. Well-induced and highly soluble recombinant proteins : 80% success

Alternative Expression Systems

Baculovirus expression system - EGFP expressed in baculovirus transfected insect cell Bright filed UV merged

Cell-free expression system Yeast expression system 1 2 3 1 2 3 4 5 6 1: Negative control 2: Positive control (GFP) 3: Hpps component II 4: Hyaluronan synthase 5: Rubber prenyl transferase 6: Marker 1: Marker 2: N3D TPL-2 using horseshoe crab signal peptide 3: N3D TPL-2 using pichia signal peptide

服務項目介紹 http://proteome.sinica.edu.tw/prod_services_01.asp 服務 編號 服務名稱 規格 收費 (台幣) D1-1 水溶性重組蛋白質之表達篩選(大腸桿菌系統) Transformed E coli. strain 14,000 D1-2 水溶性重組蛋白質之表達篩選(大腸桿菌系統)技術轉移 依需求訂定 D1-3 酵母菌系統之重組蛋白表達篩選 Pichia system 27,500 D1-4 無細胞之重組蛋白表達篩選 (使用本系統專用載體) Cell free system 18,500 D1-5 (自備質體) Cell free system (自備質體) 7,300 D1-6 桿狀病毒系統之重組蛋白表達篩選 Baculovirus expression system 36,300 http://proteome.sinica.edu.tw/prod_services_01.asp

SYSTEMS Advantages Disadvantages E. coli (14,000 NT$) Parallel cloning Fast Ease of use Low cost Poor expression Low solubility Lacking post-translational modifications Cell-free (18,500/7,300 NT$) Faster Skips cell transformation, growing, and lysis Low protein yield Expensive Tricky to optimize the lysate and expression conditions Yeast (27,500 NT$) Glycosylation Efficient Economical Protein with disulfide bonds Different glycosylation to mammalian cells Baculovirus (36,300 NT$) Most proper eukaryotic Duration of expression limited to infection period Virus production contains numerous steps Maintain high virus titers Mammalian cells Native environment for mammalian proteins Lower protein yield

Flow chart of protein production Service Requested Parallel Cloning Expression test in E. coli additional charge standard Insoluble / posttranslational modification required Soluble Yeast system Baculovirus system in vitro expression systems Protein Purification Protease cleavage to remove tag

Self-cleavage of fusion protein in vivo using TEV protease to yield native protein

-challenge to fusion protein method separation of passenger target protein from the fusion carrier fusion carriers cannot be processed by proteolysis cleaved products aggregate immediately cleaved products contain extraneous a.a. residues -our approach TEVP intracellular processing system tobacco etch virus protease (TEVP) -Glu(P6)-P5-P4-Tyr(P3)-P2-Gln(P1)-  -P1'-

In vivo cleavage of fusion proteins. TEVP intracellular processing system In vivo cleavage of fusion proteins.

Different amino acid residues at the P1' position TEVP intracellular processing system Different amino acid residues at the P1' position - more effective than an intermolecular enzymatic reaction - even with Pro in the P1' position

all six vectors successfully carried out intracellular cleavage TEVP intracellular processing system all six vectors successfully carried out intracellular cleavage

TEVP intracellular processing system Summary introduce cloning sites to target genes, without using restriction enzymes. produce native proteins with original amino termini in vivo via intracellular self-cleavage skip tedious optimization of cleavage conditions